4.3.1 TRIzol® extraction
Advanced preparation: You will need RNase-free bench, pipettes, barrier tips, pestles and 1.5 ml microcentrifuge tubes. Bench tops and other glass and plastic surfaces can be treated to remove RNAse contamination by application of RNAse Zap (Ambion), following manufacturer’s protocol. Disposable tips, pestles, and microcentrifuge tubes should be purchased RNase–free. You will need cold 75-80% ethanol and 100% isopropanol, both nuclease-free; and a pre-chilled centrifuge (at 4°C for 30 min) for Step 9. Have ready at room temperature, the TRIzol® and other reagents needed. It is recommended to use a vented fume hood for safety when working with TRIzol® and chloroform. It is also very important to work quickly with bee tissue, as it is possible that RNA will degrade if bees thaw for ten or more minutes (Dainat et al., 2011).
In a very sterile (RNAase-free) environment:
- Add 500ml of TRIzol® to frozen bee abdomens in 1.5 ml tubes.
- Mash the tissue until completely homogenized
with a pestle and shaking.
All soft tissues should be disrupted completely. Remove pestle and scrape it off along the rim of the microcentrifuge tube. Sample should be viscous.
- Centrifuge at 5,000 rpm for 1 min to pellet debris.
- Transfer the TRIzol® suspension to a fresh tube, leaving out the chitinous debris pellet.
- Add another 500ml TRIzol® and invert several times to mix.
- Add 200ml chloroform.
- Shake vigorously for 15 sec.
Do not vortex! This may increase DNA contamination in your sample.
- Incubate at RT for 2-3 min.
- Spin at 4°C for 15 min at ~14,000 rpm.
NOTE: Be especially diligent about avoiding RNases from this point on!
- Label a fresh set of RNase-free microcentrifuge tubes.
- Carefully remove tubes from centrifuge.
- Use a 1ml pipette tip with pipettor set at 550ml to draw off the upper phase and transfer it to a fresh tube.
Carefully avoid the interface (one product that ensures a clean break between phases is the Phaselock gel (5 Prime Inc.) and could be used here).
- Add 500ml 100% Isopropanol.
- Invert 3-5 times to mix gently.
- Incubate at RT for 10 min.
- Centrifuge at 4°C for 10 min at full speed (~12,500rpm), placing all tubes in the same rotation (e.g., hinge facing away from arc) so pellet location will be consistent.
- Carefully siphon off liquid using a 1ml
Observe the pellet (white) so you do not inadvertently aspirate it into the tip! Be cautious as it may dislodge and float.
- Add 1ml of cold 75-80% nuclease-free EtOH.
- Invert several times to mix.
- Spin at 4°C for 5 min at full speed.
- Carefully decant liquid using a 1ml pipette tip, avoiding the pellet and tilting the tube so no alcohol remains at the bottom of the tube covering the pellet.
- Let tubes air dry in a clean area just until the EtOH has evaporated (~20-30 min).
- Resuspend RNA pellet in 100ml of RNase-free water.
- Incubate at 55°-60°C for 10 min in water bath, ideally with shaking or flicking tubes for 10 seconds once during this time.
- Quantify and validate RNA integrity using spectrophotometer (Nanodrop, section 3.2.1), following manufacturer’s protocols, or run a small amount on 1-2% agarose gel (see section 3.2.1) to verify RNA quality. This can be accomplished by looking for degradation products migrating as a diffuse smear below the sharp 28S and 18S ribosomal RNA bands, which migrate at an analogous rate to ca. 1.75 and 2 kb double-stranded DNA markers. Alternatively, an Agilent 2100 RNA chip will provide both an accurate quantification and a measure of RNA integrity.
- Freeze for storage at -80°C for long term storage, -20°C if you plan to use the RNA within 24hrs.
- Yields should be at least 100 mg (1 mg/ml) total RNA.